WO2021248531A1 - Nucleic acid test kit - Google Patents

Nucleic acid test kit Download PDF

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Publication number
WO2021248531A1
WO2021248531A1 PCT/CN2020/096723 CN2020096723W WO2021248531A1 WO 2021248531 A1 WO2021248531 A1 WO 2021248531A1 CN 2020096723 W CN2020096723 W CN 2020096723W WO 2021248531 A1 WO2021248531 A1 WO 2021248531A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
storage component
compartment
isolation valve
fluid isolation
Prior art date
Application number
PCT/CN2020/096723
Other languages
French (fr)
Chinese (zh)
Inventor
王虎
李永峰
张新建
胡在兵
张国豪
Original Assignee
北京百康芯生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京百康芯生物科技有限公司 filed Critical 北京百康芯生物科技有限公司
Priority to KR1020217023946A priority Critical patent/KR20210154799A/en
Priority to EP20916238.7A priority patent/EP3943589A4/en
Priority to US17/426,637 priority patent/US20220235407A1/en
Priority to JP2021549907A priority patent/JP2022540738A/en
Publication of WO2021248531A1 publication Critical patent/WO2021248531A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502738—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by integrated valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/04—Exchange or ejection of cartridges, containers or reservoirs
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/16—Reagents, handling or storing thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/046—Function or devices integrated in the closure
    • B01L2300/048—Function or devices integrated in the closure enabling gas exchange, e.g. vents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0636—Integrated biosensor, microarrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0672—Integrated piercing tool
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0877—Flow chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/12—Specific details about materials
    • B01L2300/123—Flexible; Elastomeric
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0478—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure pistons
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0655—Valves, specific forms thereof with moving parts pinch valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0677—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
    • B01L2400/0683—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers mechanically breaking a wall or membrane within a channel or chamber

Definitions

  • the invention relates to a nucleic acid detection cassette, in particular to a nucleic acid detection cassette.
  • the present invention provides a nucleic acid detection cartridge.
  • the present invention provides a nucleic acid detection cartridge, comprising a substrate, a liquid storage component, a solid reagent storage component, and an amplification reaction zone, the substrate is connected to the amplification reaction zone, the liquid storage component, and the solid reagent
  • the storage components are respectively arranged on the substrate, the liquid storage component and the solid reagent storage component are communicated with each other through a micro flow channel, and the solid reagent storage component is communicated with the amplification reaction zone through a micro flow channel.
  • the liquid storage component includes a sample storage component, an extraction reagent storage component, and a waste liquid fixing chamber
  • the substrate is provided with a nucleic acid fixation extraction reaction cell
  • the sample storage component, and the extraction reagent storage component The output ends of the nucleic acid fixation extraction reaction cell are respectively communicated with the input end of the nucleic acid fixation extraction reaction cell through a micro flow channel, and the output ends of the nucleic acid fixation extraction reaction cell are respectively communicated with the solid reagent storage assembly and the waste liquid fixation chamber through the micro flow channel.
  • the extraction reagent storage assembly includes a plurality of independent compartments, the compartments including a tube wall, a plunger and a diaphragm, the plunger is arranged above the tube wall, and the diaphragm Is arranged below the tube wall, the tube wall, the plunger, and the diaphragm are enclosed to form a liquid reagent storage cavity, the substrate is provided with sharp protrusions corresponding to the compartments one to one, the The sharp protrusion is arranged under the diaphragm.
  • the compartments include at least a first compartment for storing binding liquid, a second compartment for storing rinsing liquid, and a third compartment for storing eluent.
  • the channel is in communication with the sample storage component, the microchannel between the sample storage component and the nucleic acid fixation extraction reaction cell is provided with a first fluid isolation valve, and between the second compartment and the nucleic acid fixation extraction reaction cell A second fluid isolation valve is provided on the micro flow channel, a third fluid isolation valve is provided on the micro flow channel between the third compartment and the nucleic acid fixation extraction reaction cell, and the nucleic acid fixation extraction reaction cell is connected to the solid A fourth fluid isolation valve is provided on the micro flow channel between the reagent storage components, and a fifth fluid isolation valve is provided on the micro flow channel between the nucleic acid fixation extraction reaction cell and the waste liquid fixation chamber.
  • the solid reagent A sixth fluid isolation valve is provided on the micro flow channel between the storage component and the amplification reaction zone.
  • the compartment further includes a pre-amplification reagent compartment for storing pre-amplification reagents, the pre-amplification reagent compartment is connected to the nucleic acid fixed extraction reaction cell through a micro flow channel, and the A seventh fluid isolation valve is provided on the micro flow channel between the pre-amplification reagent compartment and the nucleic acid fixation extraction reaction cell.
  • a plurality of interconnected grooves are provided on the substrate, a cover sheet is attached to the substrate, and the cover sheet encloses the grooves to form a micro flow channel.
  • a plurality of valve seat structures are provided on the chip, and the plurality of valve seat structures are respectively installed with a first fluid isolation valve, a second fluid isolation valve, a third fluid isolation valve, a fourth fluid isolation valve, a fifth fluid isolation valve, and a sixth fluid isolation valve. Fluid isolation valve.
  • the waste liquid fixing chamber is provided with a first airway interface that can be independently controlled to open and close and a second airway interface that can be independently controlled to open and close.
  • the sample storage assembly is connected to the first airway interface.
  • An airway interface is in communication, and the third compartment or the pre-amplification reagent compartment is in communication with the second airway interface.
  • the sample storage assembly is provided with a first magnetic rotor
  • the nucleic acid immobilization extraction reaction cell is provided with a second magnetic rotor
  • the sample storage assembly is installed to drive the first magnetic rotor to rotate.
  • the first magnet rotation mechanism of the nucleic acid fixation extraction reaction cell is equipped with a second magnet rotation mechanism that drives the second magnetic rotor to rotate
  • the nucleic acid fixation extraction reaction pool is equipped with a first heating module
  • the expansion A second heating module is installed at the increasing reaction zone.
  • the sample storage component is mainly composed of a chamber and a cover, and the nucleic acid capture magnetic beads required in the nucleic acid extraction process are pre-stored in the interior of the chamber of the sample storage component.
  • the waste liquid fixing chamber contains a porous water-absorbing material.
  • the amplification reaction zone is mainly composed of a plurality of reaction holes and pipes connecting the reaction holes, the amplification reaction zone is connected to the substrate through a connecting piece, and the amplification reaction zone is above Fluorescence imaging processing module is installed.
  • the beneficial effects of the present invention are: through the above solution, all reagents required for nucleic acid extraction and amplification can be built in the integrated cartridge, liquid reagents are stored in the liquid storage component, and dry powder reagents are stored in the solid reagent storage component. , The user only needs to add samples, the operation is extremely simple, and it is really suitable for basic users.
  • Figure 1 is an overall schematic diagram of a nucleic acid detection cartridge of the present invention.
  • Figure 2 is a schematic diagram of a nucleic acid detection cartridge of the present invention.
  • Fig. 3 is a schematic diagram of the structure of a compartment of a nucleic acid detection cartridge of the present invention.
  • Figure 4 is a schematic diagram of a nucleic acid detection cartridge of the present invention with a pre-amplification reagent compartment added.
  • the present invention provides a nucleic acid detection cartridge, which is an integrated cartridge, including a substrate 1, a liquid storage component 2, a solid reagent storage component 3, an amplification reaction zone 4.
  • the above-mentioned components are assembled and bonded to form a sealed integrated cartridge composed of multiple interconnected areas.
  • the substrate 1 and the The amplification reaction zone 4 is connected, the liquid storage component 2 and the solid reagent storage component 3 are respectively arranged on the substrate 1, and the liquid storage component 2 and the solid reagent storage component 3 are in communication with each other through a micro channel, so The solid reagent storage assembly 3 and the amplification reaction zone 4 are in communication with each other through a micro flow channel.
  • the liquid storage component 2 includes a sample storage component 201, an extraction reagent storage component 202, and a waste liquid fixing chamber 203.
  • the substrate 1 is provided with a nucleic acid fixing extraction reaction cell 102, so The output ends of the sample storage component 201 and the extraction reagent storage component 202 are respectively connected to the input end of the nucleic acid immobilization extraction reaction cell 102 through a micro flow channel, and the output ends of the nucleic acid immobilization extraction reaction cell 102 are respectively connected to the input end of the nucleic acid immobilization extraction reaction cell 102 through the micro flow channel.
  • the solid reagent storage component 3 and the waste liquid fixing chamber 203 are in communication.
  • the solid reagent storage component includes a storage cylinder 301 and a connection structure.
  • the fixed reagent storage component 3 contains reagents for nucleic acid amplification, such as dNTP, Taq polymerase, etc., pre-stored in the form of dry powder. 301 ⁇ Cylinder 301.
  • the extraction reagent storage assembly 202 includes a plurality of independent compartments.
  • the compartments include a tube wall 2021, a plunger 2022, and a diaphragm 2023. Above the wall 2021, the diaphragm 2023 is arranged below the tube wall 2021, the tube wall 2021, the plunger 2022, the diaphragm 2023 are enclosed to form a liquid reagent storage cavity, and the substrate 1 is provided with
  • the sharp protrusions 103 are one-to-one corresponding to the compartments, and the sharp protrusions 103 are arranged below the diaphragm 2025 and used to pierce the diaphragm 2025 to release the liquid reagent in the liquid reagent storage cavity.
  • the compartment includes at least a first compartment 202a for storing binding liquid, a second compartment 202b for storing rinsing liquid, and a third compartment 202c for storing eluent.
  • the compartment 202a is in communication with the sample storage component 201 through a microchannel.
  • a first fluid isolation valve 701 is provided on the microchannel between the sample storage component 201 and the nucleic acid immobilization extraction reaction cell 102, and the second fluid isolation valve 701 is provided on the microchannel.
  • a second fluid isolation valve 702 is provided on the micro flow channel between the compartment 202b and the nucleic acid fixation extraction reaction cell 102, and the micro flow channel between the third compartment 202c and the nucleic acid fixation extraction reaction cell 102 is provided There is a third fluid isolation valve 703, a fourth fluid isolation valve 704 is provided on the microchannel between the nucleic acid fixation extraction reaction cell 102 and the solid reagent storage assembly 3, and the nucleic acid fixation extraction reaction cell 102 is connected to the A fifth fluid isolation valve 705 is provided on the micro flow channel between the waste liquid fixing chambers 203, and a sixth fluid isolation valve 706 is provided on the micro flow channel between the solid reagent storage assembly 3 and the amplification reaction zone 4 .
  • the compartment also includes a pre-amplification reagent compartment 202d storing pre-amplification reagents, and the pre-amplification reagent compartment 202d is connected to the nucleic acid immobilization extraction reaction cell through a micro flow channel.
  • the micro flow channel between the pre-amplification reagent compartment 202d and the nucleic acid fixation extraction reaction cell 102 is provided with a seventh fluid isolation valve 707, and the pre-amplification reagent compartment can also be added according to the test procedure 202d pre-stores other necessary liquid reagents, such as nucleic acid amplification reagents used in the first step of nested amplification, including target probes, specific primers, dNTPs, Taq polymerase, reaction buffers, etc. .
  • the substrate 3 is provided with a plurality of interconnected grooves 101, the substrate 1 is attached with a cover sheet 5, and the cover sheet 5 connects the grooves 101 Enclosed to form a micro flow channel, after the cover sheet 5 is attached to the two sides of the substrate 1, a plurality of closed interconnected pipes and chambers are formed.
  • the substrate 1 is provided with a plurality of valve seat structures 105, The valve seat structure 105 is equipped with a plurality of fluid isolation valves 7 respectively.
  • the waste liquid fixing chamber 203 is provided with a first airway interface 1071 that can be independently controlled to open and close and a second airway interface 1072 that can be independently controlled to open and close.
  • the sample storage The component 201 is in communication with the first airway interface 1071, and the third compartment 202c or the pre-amplification reagent compartment 202d is in communication with the second airway interface 1072.
  • the rotor part 6 includes a first magnetic rotor 601 and a second magnetic rotor 602.
  • the sample storage assembly 201 is provided with a first magnetic rotor 601
  • the nucleic acid immobilization extraction reaction cell 102 is provided with a first magnetic rotor 601.
  • There is a second magnetic rotor 602 a first magnet rotating mechanism that drives the first magnetic rotor 601 is installed at the sample storage assembly 201, and the second magnetic rotor is installed at the nucleic acid fixed extraction reaction cell 102.
  • the first magnetic rotor 601 is a mixing cell rotor
  • the second magnetic rotor 602 is a fixed extraction reaction cell rotor
  • the nucleic acid fixed extraction reaction cell 102 is equipped with a first heating module
  • the expansion A second heating module is installed in the reaction zone 4.
  • the sample storage component 201 is mainly composed of a chamber 2011 and a lid.
  • the chamber 2011 of the sample storage component 201 is pre-stored with nucleic acid capture magnetic beads used in the nucleic acid extraction process. .
  • the waste liquid fixing chamber 203 contains a set of porous water-absorbing materials, such as sponge and/or absorbent paper, and a waste liquid outlet.
  • the amplification reaction zone 4 is mainly composed of a plurality of reaction holes 401 and a pipe 402 connecting the reaction holes 401, and the amplification reaction zone 4 is connected to the substrate 1 through a connector.
  • a fluorescence imaging processing module is installed above the amplification reaction zone 4.
  • the base sheet 1 and the liquid storage component 2, the fixed reagent storage component 3, and the cover sheet 5 are attached together using one or more of methods such as buckle, gluing, and hot pressing. .
  • the reaction wells 401 in the amplification reaction zone 4 are preliminarily stored in a dry form with target probes or primers for nucleic acid amplification.
  • the substrate 1, the liquid storage component 2, the solid reagent storage component 3, the amplification reaction zone 4, and the cover sheet 5, the main body is made of high molecular polymer, which can be polycarbonate or polycarbonate.
  • high molecular polymer which can be polycarbonate or polycarbonate.
  • the fluid isolation valve 7 is made of an elastic material with good sealing performance, which can be one or more of natural rubber, silica gel, nitrile rubber, butyl rubber, fluorine rubber, and ethylene propylene rubber. .
  • the shell is made of high molecular polymer, which can be polycarbonate, polymethyl methacrylate, cyclic olefin copolymer, polypropylene, polyethylene terephthalate, acrylonitrile -One or more of butadiene-styrene copolymers.
  • Example 1 of the use of a nucleic acid detection cartridge provided by the present invention is as follows:
  • the external source instrument includes at least 2 airway ports that can be turned on and off independently (the first airway port 1071, the second airway port 1072), 10 pressure rods, 2 local heating modules, and 2 containing magnets Rotation mechanism (first magnet rotation mechanism, second magnet rotation mechanism), and a fluorescence imaging processing module, each fluid isolation valve 7 is correspondingly installed with a down lever that controls its switch, and a plunger in each compartment
  • Each 2022 corresponds to the installation of a pressing rod to control its pressing down.
  • a plurality of lower pressure rods separate the first fluid isolation valve 701, the second fluid isolation valve 702, the third fluid isolation valve 703, and the fourth fluid respectively.
  • the valve 704 is closed and the first airway interface 1071 is closed.
  • the chamber 2011 forms a closed and independent space. Turning on the first magnet rotation mechanism on the external instrument located below the chamber 2011 can achieve a sufficient mixing reaction of the sample and the embedded reagent. The purpose of this step is to release the nucleic acid to be detected in the sample.
  • the first magnetic rotor 601 will fully mix the released nucleic acid, magnetic beads and binding liquid, and then open the first fluid isolation valve 701, and at the same time the gas source passes through the first airway interface 1071 to the cavity Positive pressure is provided in the chamber 2011 to push the above-mentioned mixed liquid, in accordance with the "chamber 2011-first fluid isolation valve 701 (open)-nucleic acid immobilization extraction reaction cell 102-fourth fluid isolation valve 704 (closed)-fifth fluid isolation valve 705 (open)-the waste liquid is flowed in the order of the fixed chamber 203".
  • the lower pressing rod located on the second compartment 202b descends, pushing the plunger 2022 to press the diaphragm 2023 to expand and deform, and is pierced by the sharp protrusion 103 on the substrate 1, releasing the nucleic acid rinsing liquid in the second compartment 202b.
  • the lower pressing rod can press out the liquid in the compartment at one time, or by controlling the descending height, the liquid can be released quantitatively in batches for multiple rinsing.
  • the nucleic acid rinsing solution released from the second compartment 202b enters the nucleic acid fixation extraction reaction tank 102 through the second fluid isolation valve 702.
  • the nucleic acid fixation extraction reaction tank 102 is a chamber with a low aspect ratio, and its projection shape can be circular , Diamond-shaped, olive-shaped, gourd-shaped, etc., the above-mentioned various shapes ensure that the liquid can fill the cavity.
  • the second magnet rotating mechanism below starts to move, driving the second magnetic rotor 602 to fully mix the magnetic beads fixed in the chamber in the previous step with the rinsing liquid.
  • the second magnet rotation mechanism stops moving, and after being allowed to stand for a period of time under the action of magnetic force, the magnetic bead particles suspended in the chamber are fixed in the nucleic acid immobilization extraction reaction cell 102 again.
  • the lower pressing rod located on the third compartment 203 descends, pushes the plunger 2032 to press the diaphragm 2033 to expand and deform, and is pierced by the sharp protrusion 103 on the substrate 1
  • the nucleic acid eluate in the third compartment 202c is released.
  • the nucleic acid eluate released from the third compartment 202c enters the nucleic acid fixation extraction reaction cell 102 through the third fluid isolation valve 703.
  • the second magnet rotating mechanism below starts to move, driving the second magnetic rotor 602 to fully mix the magnetic beads fixed in the chamber in the previous step with the eluent, and at the same time turn on the nucleic acid
  • the first heating module under the extraction reaction tank 102 is fixed.
  • the temperature of "mixing + heating" is controlled at 50-80°C, and the time is controlled at 180-600s, the effect is the best.
  • the lower pressing rod located above the storage cylinder 301 descends, pushing the mixed reagents into the reaction hole 401 in the amplification reaction zone 4.
  • each reaction hole 401 is isolated from the outside, the second heating module located below the amplification reaction zone 4 is turned on, and the fluorescence imaging process located above the amplification reaction zone 4 is turned on Module.
  • the reaction temperature is controlled, and each independent reaction hole starts a nucleic acid amplification reaction, and the fluorescence imaging processing module detects the target gene according to the light signal of the reaction hole.
  • the first stage operation of this embodiment is basically the same as steps 1-13 of embodiment 1, that is, the nucleic acid eluate in the third compartment 202c is injected into the nucleic acid fixation extraction reaction cell 102, but the corresponding plunger is controlled
  • the descent distance of 2032 makes the eluate not completely fill the nucleic acid fixation extraction reaction cell 102;
  • the amplification reaction may be isothermal nucleic acid amplification or variable temperature nucleic acid amplification.
  • part of the amplified product passes through the fourth fluid isolation valve 704 and the fifth fluid isolation valve 705 Enter the waste liquid fixing chamber 203.
  • the remaining amount can be 1/10-1/100 of the total nucleic acid fixation extraction reaction tank 102, or other suitable ratios required by the actual amplification reaction.
  • the diluted pre-amplification product enters the solid reagent storage assembly from the nucleic acid immobilization extraction reaction cell 102 3 of the storage cylinder 301, and mixed with the reagents in it.

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Abstract

Provided is a nucleic acid test kit, comprising a substrate, a liquid storage component, a solid reagent storage component and an amplification reaction region, wherein the substrate is connected to the amplification reaction region; the liquid storage component and the solid reagent storage component are separately arranged on the substrate; the liquid storage component is in communication with the solid reagent storage component by means of a micro-channel; and the solid reagent storage component is in communication with the amplification reaction region by means of a micro-channel. The present invention has the beneficial effect that all reagents required by nucleic acid extraction and amplification can be internally placed into an integrated kit, with liquid reagents being stored in the liquid storage component, and dry powder reagents being stored in the solid reagent storage component, such that a user only needs to add a sample, and the operation is very simple and is suitable for community-level users.

Description

一种核酸检测卡盒Nucleic acid detection cassette 技术领域Technical field
本发明涉及核酸检测盒，尤其涉及一种核酸检测卡盒。The invention relates to a nucleic acid detection cassette, in particular to a nucleic acid detection cassette.
背景技术Background technique
现有核酸检测盒的集成度较低，使用较为复杂，还需要用户手工加入部分试剂，经常误操作，导致检测结果不准确，不适用于基层用户使用。Existing nucleic acid detection kits have low integration and are more complicated to use. Users also need to manually add some reagents. Frequent misoperations result in inaccurate detection results and are not suitable for use by grassroots users.
发明内容Summary of the invention
为了解决现有技术中的问题，本发明提供了一种核酸检测卡盒。In order to solve the problems in the prior art, the present invention provides a nucleic acid detection cartridge.
本发明提供了一种核酸检测卡盒，包括基片、液体存储组件、固体试剂存储组件、扩增反应区，所述基片与所述扩增反应区连接，所述液体存储组件、固体试剂存储组件分别设置在所述基片上，所述液体存储组件与所述固体试剂存储组件通过微流道连通，所述固体试剂存储组件与所述扩增反应区通过微流道连通。The present invention provides a nucleic acid detection cartridge, comprising a substrate, a liquid storage component, a solid reagent storage component, and an amplification reaction zone, the substrate is connected to the amplification reaction zone, the liquid storage component, and the solid reagent The storage components are respectively arranged on the substrate, the liquid storage component and the solid reagent storage component are communicated with each other through a micro flow channel, and the solid reagent storage component is communicated with the amplification reaction zone through a micro flow channel.
作为本发明的进一步改进，所述液体存储组件包括样本存储组件、提取试剂存储组件、废液固定腔室，所述基片上设有核酸固定萃取反应池，所述样本存储组件、提取试剂存储组件的输出端分别通过微流道与所述核酸固定萃取反应池的输入端连通，所述核酸固定萃取反应池的输出端分别通过微流道与所述固体试剂存储组件、废液固定腔室连通。As a further improvement of the present invention, the liquid storage component includes a sample storage component, an extraction reagent storage component, and a waste liquid fixing chamber, the substrate is provided with a nucleic acid fixation extraction reaction cell, the sample storage component, and the extraction reagent storage component The output ends of the nucleic acid fixation extraction reaction cell are respectively communicated with the input end of the nucleic acid fixation extraction reaction cell through a micro flow channel, and the output ends of the nucleic acid fixation extraction reaction cell are respectively communicated with the solid reagent storage assembly and the waste liquid fixation chamber through the micro flow channel. .
作为本发明的进一步改进，所述提取试剂存储组件包括多个独立的隔间，所述隔间包括管壁、柱塞和隔膜，所述柱塞设置在所述管壁的上方，所述隔膜设置在所述管壁的下方，所述管壁、柱塞、隔膜之间围合形成液体试剂存储腔体，所述基片上设有与所述隔间一一对应的尖锐凸起，所述尖锐凸起设置在所述隔膜的下方。As a further improvement of the present invention, the extraction reagent storage assembly includes a plurality of independent compartments, the compartments including a tube wall, a plunger and a diaphragm, the plunger is arranged above the tube wall, and the diaphragm Is arranged below the tube wall, the tube wall, the plunger, and the diaphragm are enclosed to form a liquid reagent storage cavity, the substrate is provided with sharp protrusions corresponding to the compartments one to one, the The sharp protrusion is arranged under the diaphragm.
作为本发明的进一步改进，所述隔间至少包括存储结合液的第一隔间、存储漂洗液的第二隔间和存储洗脱液的第三隔间，所述第一隔间通过微流道与所述样本存储组件连通，所述样本存储组件与所述核酸固定萃取反应池之间的微流道上设有第一流体隔离阀，所述第二隔间与所述核酸固定萃取反应池之间的微流道上设有第二流体隔离阀，所述第三隔间与所述核酸固定萃取反应池之间的微流道上设有第三流体隔离阀，所述核酸固定萃取反应池与所述固体试剂存储组件之间的微流道上设有第四流体隔离阀，所述核酸固定萃取反应池与所述废液固定腔室之间的微流道上设有第五流体隔离阀，所述固体试剂存储组件与所述扩增反应区之间的微流道上设有第 六流体隔离阀。As a further improvement of the present invention, the compartments include at least a first compartment for storing binding liquid, a second compartment for storing rinsing liquid, and a third compartment for storing eluent. The channel is in communication with the sample storage component, the microchannel between the sample storage component and the nucleic acid fixation extraction reaction cell is provided with a first fluid isolation valve, and between the second compartment and the nucleic acid fixation extraction reaction cell A second fluid isolation valve is provided on the micro flow channel, a third fluid isolation valve is provided on the micro flow channel between the third compartment and the nucleic acid fixation extraction reaction cell, and the nucleic acid fixation extraction reaction cell is connected to the solid A fourth fluid isolation valve is provided on the micro flow channel between the reagent storage components, and a fifth fluid isolation valve is provided on the micro flow channel between the nucleic acid fixation extraction reaction cell and the waste liquid fixation chamber. The solid reagent A sixth fluid isolation valve is provided on the micro flow channel between the storage component and the amplification reaction zone.
作为本发明的进一步改进，所述隔间还包括存储预扩增试剂的预扩增试剂隔间，所述预扩增试剂隔间通过微流道与所述核酸固定萃取反应池连通，所述预扩增试剂隔间与所述核酸固定萃取反应池之间的微流道上设有第七流体隔离阀。As a further improvement of the present invention, the compartment further includes a pre-amplification reagent compartment for storing pre-amplification reagents, the pre-amplification reagent compartment is connected to the nucleic acid fixed extraction reaction cell through a micro flow channel, and the A seventh fluid isolation valve is provided on the micro flow channel between the pre-amplification reagent compartment and the nucleic acid fixation extraction reaction cell.
作为本发明的进一步改进，所述基片上设有多条相互连通的沟槽，所述基片上贴合有盖片，所述盖片将所述沟槽围合形成微流道，所述基片上设有多个阀座结构，多个所述阀座结构分别安装第一流体隔离阀、第二流体隔离阀、第三流体隔离阀、第四流体隔离阀、第五流体隔离阀、第六流体隔离阀。As a further improvement of the present invention, a plurality of interconnected grooves are provided on the substrate, a cover sheet is attached to the substrate, and the cover sheet encloses the grooves to form a micro flow channel. A plurality of valve seat structures are provided on the chip, and the plurality of valve seat structures are respectively installed with a first fluid isolation valve, a second fluid isolation valve, a third fluid isolation valve, a fourth fluid isolation valve, a fifth fluid isolation valve, and a sixth fluid isolation valve. Fluid isolation valve.
作为本发明的进一步改进，所述废液固定腔室上设有可独立控制开启关闭的第一气道接口和可独立控制开启关闭的第二气道接口，所述样本存储组件与所述第一气道接口连通，所述第三隔间或者预扩增试剂隔间与所述第二气道接口连通。As a further improvement of the present invention, the waste liquid fixing chamber is provided with a first airway interface that can be independently controlled to open and close and a second airway interface that can be independently controlled to open and close. The sample storage assembly is connected to the first airway interface. An airway interface is in communication, and the third compartment or the pre-amplification reagent compartment is in communication with the second airway interface.
作为本发明的进一步改进，所述样本存储组件内设有第一磁力转子，所述核酸固定萃取反应池内设有第二磁力转子，所述样本存储组件处安装有驱动所述第一磁力转子转动的第一磁铁旋转机构，所述核酸固定萃取反应池处安装有驱动所述第二磁力转子转动的第二磁铁旋转机构，所述核酸固定萃取反应池处安装有第一加热模块，所述扩增反应区处安装有第二加热模块。As a further improvement of the present invention, the sample storage assembly is provided with a first magnetic rotor, the nucleic acid immobilization extraction reaction cell is provided with a second magnetic rotor, and the sample storage assembly is installed to drive the first magnetic rotor to rotate. The first magnet rotation mechanism of the nucleic acid fixation extraction reaction cell is equipped with a second magnet rotation mechanism that drives the second magnetic rotor to rotate, the nucleic acid fixation extraction reaction pool is equipped with a first heating module, and the expansion A second heating module is installed at the increasing reaction zone.
作为本发明的进一步改进，所述样本存储组件主要由腔室和盖子组成，在所述样本存储组件的腔室内部预存有用于核酸提取流程中所需的核酸捕获磁珠。As a further improvement of the present invention, the sample storage component is mainly composed of a chamber and a cover, and the nucleic acid capture magnetic beads required in the nucleic acid extraction process are pre-stored in the interior of the chamber of the sample storage component.
作为本发明的进一步改进，所述废液固定腔室内含多孔吸水材料。As a further improvement of the present invention, the waste liquid fixing chamber contains a porous water-absorbing material.
作为本发明的进一步改进，所述扩增反应区主要由多个反应孔和连接反应孔的管道组成，所述扩增反应区通过连接件与所述基片连接，所述扩增反应区上方安装有荧光成像处理模块。As a further improvement of the present invention, the amplification reaction zone is mainly composed of a plurality of reaction holes and pipes connecting the reaction holes, the amplification reaction zone is connected to the substrate through a connecting piece, and the amplification reaction zone is above Fluorescence imaging processing module is installed.
本发明的有益效果是：通过上述方案，可以将核酸提取与扩增所需的所有试剂都内置在集成化卡盒上，将液体试剂存储在液体存储组件，将干粉试剂存储在固体试剂存储组件，用户只需要加入样品，操作极其简单，真正适用于基层用户使用。The beneficial effects of the present invention are: through the above solution, all reagents required for nucleic acid extraction and amplification can be built in the integrated cartridge, liquid reagents are stored in the liquid storage component, and dry powder reagents are stored in the solid reagent storage component. , The user only needs to add samples, the operation is extremely simple, and it is really suitable for basic users.
附图说明Description of the drawings
图1是本发明一种核酸检测卡盒的整体示意图。Figure 1 is an overall schematic diagram of a nucleic acid detection cartridge of the present invention.
图2是本发明一种核酸检测卡盒的示意图。Figure 2 is a schematic diagram of a nucleic acid detection cartridge of the present invention.
图3是本发明一种核酸检测卡盒的隔间的结构示意图。Fig. 3 is a schematic diagram of the structure of a compartment of a nucleic acid detection cartridge of the present invention.
图4是本发明一种核酸检测卡盒增加预扩增试剂隔间的示意图。Figure 4 is a schematic diagram of a nucleic acid detection cartridge of the present invention with a pre-amplification reagent compartment added.
具体实施方式detailed description
下面结合附图说明及具体实施方式对本发明作进一步说明。The present invention will be further described below in conjunction with the description of the drawings and the specific embodiments.
如图1至图4所示，本发明提供了一种核酸检测卡盒，是一种集成化卡盒，包括基片1、液体存储组件2、固体试剂存储组件3、扩增反应区4、盖片5、转子零件6、流体隔离阀7、外壳，上述组件经过装配和贴合之后，形成由多条相互连通区域的所组成的密封的集成化卡盒，所述基片1与所述扩增反应区4连接，所述液体存储组件2、固体试剂存储组件3分别设置在所述基片1上，所述液体存储组件2与所述固体试剂存储组件3通过微流道连通，所述固体试剂存储组件3与所述扩增反应区4通过微流道连通。As shown in Figures 1 to 4, the present invention provides a nucleic acid detection cartridge, which is an integrated cartridge, including a substrate 1, a liquid storage component 2, a solid reagent storage component 3, an amplification reaction zone 4. The cover sheet 5, the rotor part 6, the fluid isolation valve 7, and the housing. The above-mentioned components are assembled and bonded to form a sealed integrated cartridge composed of multiple interconnected areas. The substrate 1 and the The amplification reaction zone 4 is connected, the liquid storage component 2 and the solid reagent storage component 3 are respectively arranged on the substrate 1, and the liquid storage component 2 and the solid reagent storage component 3 are in communication with each other through a micro channel, so The solid reagent storage assembly 3 and the amplification reaction zone 4 are in communication with each other through a micro flow channel.
如图1至图4所示，所述液体存储组件2包括样本存储组件201、提取试剂存储组件202、废液固定腔室203，所述基片1上设有核酸固定萃取反应池102，所述样本存储组件201、提取试剂存储组件202的输出端分别通过微流道与所述核酸固定萃取反应池102的输入端连通，所述核酸固定萃取反应池102的输出端分别通过微流道与所述固体试剂存储组件3、废液固定腔室203连通。As shown in Figures 1 to 4, the liquid storage component 2 includes a sample storage component 201, an extraction reagent storage component 202, and a waste liquid fixing chamber 203. The substrate 1 is provided with a nucleic acid fixing extraction reaction cell 102, so The output ends of the sample storage component 201 and the extraction reagent storage component 202 are respectively connected to the input end of the nucleic acid immobilization extraction reaction cell 102 through a micro flow channel, and the output ends of the nucleic acid immobilization extraction reaction cell 102 are respectively connected to the input end of the nucleic acid immobilization extraction reaction cell 102 through the micro flow channel. The solid reagent storage component 3 and the waste liquid fixing chamber 203 are in communication.
如图1至图4所示，固体试剂存储组件包括存储筒301、连接结构，固定试剂存储组件3包含用于核酸扩增的试剂，如dNTP，Taq聚合酶等，以干粉的形式预存在存储筒301中。As shown in Figures 1 to 4, the solid reagent storage component includes a storage cylinder 301 and a connection structure. The fixed reagent storage component 3 contains reagents for nucleic acid amplification, such as dNTP, Taq polymerase, etc., pre-stored in the form of dry powder. 301中。 Cylinder 301.
如图1至图4所示，所述提取试剂存储组件202包括多个独立的隔间，所述隔间包括管壁2021、柱塞2022和隔膜2023，所述柱塞2022设置在所述管壁2021的上方，所述隔膜2023设置在所述管壁2021的下方，所述管壁2021、柱塞2022、隔膜2023之间围合形成液体试剂存储腔体，所述基片1上设有与所述隔间一一对应的尖锐凸起103，所述尖锐凸起103设置在所述隔膜2025的下方，用于刺空隔膜2025，以释放液体试剂存储腔体内的液体试剂。As shown in FIGS. 1 to 4, the extraction reagent storage assembly 202 includes a plurality of independent compartments. The compartments include a tube wall 2021, a plunger 2022, and a diaphragm 2023. Above the wall 2021, the diaphragm 2023 is arranged below the tube wall 2021, the tube wall 2021, the plunger 2022, the diaphragm 2023 are enclosed to form a liquid reagent storage cavity, and the substrate 1 is provided with The sharp protrusions 103 are one-to-one corresponding to the compartments, and the sharp protrusions 103 are arranged below the diaphragm 2025 and used to pierce the diaphragm 2025 to release the liquid reagent in the liquid reagent storage cavity.
如图1至图4所示，所述隔间至少包括存储结合液的第一隔间202a、存储漂洗液的第二隔间202b和存储洗脱液的第三隔间202c，所述第一隔间202a通过微流道与所述样本存储组件201连通，所述样本存储组件201与所述核酸固定萃取反应池102之间的微流道上设有第一流体隔离阀701，所述第二隔间202b与所述核酸固定萃取反应池102之间的微流道上设有第二 流体隔离阀702，所述第三隔间202c与所述核酸固定萃取反应池102之间的微流道上设有第三流体隔离阀703，所述核酸固定萃取反应池102与所述固体试剂存储组件3之间的微流道上设有第四流体隔离阀704，所述核酸固定萃取反应池102与所述废液固定腔室203之间的微流道上设有第五流体隔离阀705，所述固体试剂存储组件3与所述扩增反应区4之间的微流道上设有第六流体隔离阀706。As shown in Figures 1 to 4, the compartment includes at least a first compartment 202a for storing binding liquid, a second compartment 202b for storing rinsing liquid, and a third compartment 202c for storing eluent. The compartment 202a is in communication with the sample storage component 201 through a microchannel. A first fluid isolation valve 701 is provided on the microchannel between the sample storage component 201 and the nucleic acid immobilization extraction reaction cell 102, and the second fluid isolation valve 701 is provided on the microchannel. A second fluid isolation valve 702 is provided on the micro flow channel between the compartment 202b and the nucleic acid fixation extraction reaction cell 102, and the micro flow channel between the third compartment 202c and the nucleic acid fixation extraction reaction cell 102 is provided There is a third fluid isolation valve 703, a fourth fluid isolation valve 704 is provided on the microchannel between the nucleic acid fixation extraction reaction cell 102 and the solid reagent storage assembly 3, and the nucleic acid fixation extraction reaction cell 102 is connected to the A fifth fluid isolation valve 705 is provided on the micro flow channel between the waste liquid fixing chambers 203, and a sixth fluid isolation valve 706 is provided on the micro flow channel between the solid reagent storage assembly 3 and the amplification reaction zone 4 .
如图1至图4所示，所述隔间还包括存储预扩增试剂的预扩增试剂隔间202d，所述预扩增试剂隔间202d通过微流道与所述核酸固定萃取反应池102连通，所述预扩增试剂隔间202d与所述核酸固定萃取反应池102之间的微流道上设有第七流体隔离阀707，还可以根据测试流程，增加述预扩增试剂隔间202d以预存其他所必须的液体试剂，例如巢式扩增的第一步扩增时所使用的核酸扩增试剂，包括标靶探针、特异性引物，dNTP，Taq聚合酶，反应缓冲液等。As shown in Figures 1 to 4, the compartment also includes a pre-amplification reagent compartment 202d storing pre-amplification reagents, and the pre-amplification reagent compartment 202d is connected to the nucleic acid immobilization extraction reaction cell through a micro flow channel. 102 is connected, the micro flow channel between the pre-amplification reagent compartment 202d and the nucleic acid fixation extraction reaction cell 102 is provided with a seventh fluid isolation valve 707, and the pre-amplification reagent compartment can also be added according to the test procedure 202d pre-stores other necessary liquid reagents, such as nucleic acid amplification reagents used in the first step of nested amplification, including target probes, specific primers, dNTPs, Taq polymerase, reaction buffers, etc. .
如图1至图4所示，所述基片3上设有多条相互连通的沟槽101，所述基片1上贴合有盖片5，所述盖片5将所述沟槽101围合形成微流道，盖片5与基片1的两侧贴合后，形成封闭的多条相互连通管道和腔室，所述基片1上设有多个阀座结构105，多个所述阀座结构105分别安装多个流体隔离阀7。As shown in Figures 1 to 4, the substrate 3 is provided with a plurality of interconnected grooves 101, the substrate 1 is attached with a cover sheet 5, and the cover sheet 5 connects the grooves 101 Enclosed to form a micro flow channel, after the cover sheet 5 is attached to the two sides of the substrate 1, a plurality of closed interconnected pipes and chambers are formed. The substrate 1 is provided with a plurality of valve seat structures 105, The valve seat structure 105 is equipped with a plurality of fluid isolation valves 7 respectively.
如图1至图4所示，所述废液固定腔室203上设有可独立控制开启关闭的第一气道接口1071和可独立控制开启关闭的第二气道接口1072，所述样本存储组件201与所述第一气道接口1071连通，所述第三隔间202c或者预扩增试剂隔间202d与所述第二气道接口1072连通。As shown in Figures 1 to 4, the waste liquid fixing chamber 203 is provided with a first airway interface 1071 that can be independently controlled to open and close and a second airway interface 1072 that can be independently controlled to open and close. The sample storage The component 201 is in communication with the first airway interface 1071, and the third compartment 202c or the pre-amplification reagent compartment 202d is in communication with the second airway interface 1072.
如图1至图4所示，转子零件6包括第一磁力转子601和第二磁力转子602，所述样本存储组件201内设有第一磁力转子601，所述核酸固定萃取反应池102内设有第二磁力转子602，所述样本存储组件201处安装有驱动所述第一磁力转子601转动的第一磁铁旋转机构，所述核酸固定萃取反应池102处安装有驱动所述第二磁力转子602转动的第二磁铁旋转机构，第一磁力转子601为混合池转子，第二磁力转子602为固定萃取反应池转子，所述核酸固定萃取反应池102处安装有第一加热模块，所述扩增反应区4处安装有第二加热模块。As shown in Figures 1 to 4, the rotor part 6 includes a first magnetic rotor 601 and a second magnetic rotor 602. The sample storage assembly 201 is provided with a first magnetic rotor 601, and the nucleic acid immobilization extraction reaction cell 102 is provided with a first magnetic rotor 601. There is a second magnetic rotor 602, a first magnet rotating mechanism that drives the first magnetic rotor 601 is installed at the sample storage assembly 201, and the second magnetic rotor is installed at the nucleic acid fixed extraction reaction cell 102. 602 rotating second magnet rotating mechanism, the first magnetic rotor 601 is a mixing cell rotor, the second magnetic rotor 602 is a fixed extraction reaction cell rotor, the nucleic acid fixed extraction reaction cell 102 is equipped with a first heating module, the expansion A second heating module is installed in the reaction zone 4.
如图1至图4所示，所述样本存储组件201主要由腔室2011和盖子组成，在所述样本存储组件201的腔室2011内部预存有用于核酸提取流程中所需的核酸捕获磁珠。As shown in Figures 1 to 4, the sample storage component 201 is mainly composed of a chamber 2011 and a lid. The chamber 2011 of the sample storage component 201 is pre-stored with nucleic acid capture magnetic beads used in the nucleic acid extraction process. .
如图1至图4所示，所述废液固定腔室203内含一组多孔吸水材料，比如海绵和/或吸水纸等；以及一个废液出口。As shown in FIGS. 1 to 4, the waste liquid fixing chamber 203 contains a set of porous water-absorbing materials, such as sponge and/or absorbent paper, and a waste liquid outlet.
如图1至图4所示，所述扩增反应区4主要由多个反应孔401和连接反应孔401的管道402组成，所述扩增反应区4通过连接件与所述基片1连接，所述扩增反应区4上方安装有荧光成像处理模块。As shown in Figures 1 to 4, the amplification reaction zone 4 is mainly composed of a plurality of reaction holes 401 and a pipe 402 connecting the reaction holes 401, and the amplification reaction zone 4 is connected to the substrate 1 through a connector. A fluorescence imaging processing module is installed above the amplification reaction zone 4.
如图1至图4所示，基片1与液体存储组件2、固定试剂存储组件3、盖片5使用卡扣、胶粘、热压等方式中的一种或多种，贴合在一起。As shown in Figures 1 to 4, the base sheet 1 and the liquid storage component 2, the fixed reagent storage component 3, and the cover sheet 5 are attached together using one or more of methods such as buckle, gluing, and hot pressing. .
如图1至图4所示，扩增反应区4中的反应孔401预先以干燥形式存有用于核酸扩增的标靶探针或者引物。As shown in FIGS. 1 to 4, the reaction wells 401 in the amplification reaction zone 4 are preliminarily stored in a dry form with target probes or primers for nucleic acid amplification.
如图1至图4所示，基片1、液体存储组件2、固体试剂存储组件3、扩增反应区4、盖片5，主体由高分子聚合物构成，可以是聚碳酸酯，聚甲基丙烯酸甲酯，环烯烃共聚物，聚丙烯、聚对苯二甲酸乙二醇酯中的一种或几种。As shown in Figures 1 to 4, the substrate 1, the liquid storage component 2, the solid reagent storage component 3, the amplification reaction zone 4, and the cover sheet 5, the main body is made of high molecular polymer, which can be polycarbonate or polycarbonate. One or more of methyl acrylate, cyclic olefin copolymer, polypropylene and polyethylene terephthalate.
如图1至图4所示，流体隔离阀7由密封性良好的弹性材料构成，可以是天然橡胶、硅胶、丁腈橡胶、丁基橡胶、氟橡胶、乙丙橡胶中的一种或几种。As shown in Figures 1 to 4, the fluid isolation valve 7 is made of an elastic material with good sealing performance, which can be one or more of natural rubber, silica gel, nitrile rubber, butyl rubber, fluorine rubber, and ethylene propylene rubber. .
如图1至图4所示，外壳由高分子聚合物构成，可以是聚碳酸酯，聚甲基丙烯酸甲酯，环烯烃共聚物，聚丙烯、聚对苯二甲酸乙二醇酯、丙烯腈-丁二烯-苯乙烯共聚物中的一种或几种。As shown in Figure 1 to Figure 4, the shell is made of high molecular polymer, which can be polycarbonate, polymethyl methacrylate, cyclic olefin copolymer, polypropylene, polyethylene terephthalate, acrylonitrile -One or more of butadiene-styrene copolymers.
本发明提供的一种核酸检测卡盒的使用实施例1如下：Example 1 of the use of a nucleic acid detection cartridge provided by the present invention is as follows:
实施例1——集成提取与扩增Example 1-Integrated extraction and amplification
1.现将本发明描述的核酸自动化提取并检测的集成化卡盒的实施方式描述如下；1. The implementation of the integrated cassette for automatic nucleic acid extraction and detection described in the present invention is now described as follows;
2.将液体样本(唾液、液化痰液、血液、拭子涮洗液等)加入样本存储组件201的腔室2011中，然后关闭盖子(图中未示出)，放入配套的外源设备中，即可开始进行自动化的核酸提取及检测。2. Put the liquid sample (saliva, liquefied sputum, blood, swab rinse, etc.) into the chamber 2011 of the sample storage component 201, then close the lid (not shown in the figure), and put it into the supporting external equipment In, you can start automated nucleic acid extraction and detection.
3.外源仪器包含至少2个可独立控制开启关闭的气道接口(第一气道接口1071、第二气道接口1072)、10个下压杆、2个局部加热模块、2个包含磁铁的旋转机构(第一磁铁旋转机构、第二磁铁旋转机构)，以及1个荧光成像处理模块，每个流体隔离阀7均对应安装一个控制其开关的下压杆，每个隔间的柱塞2022均对应安装一个控制其下压的下压杆。3. The external source instrument includes at least 2 airway ports that can be turned on and off independently (the first airway port 1071, the second airway port 1072), 10 pressure rods, 2 local heating modules, and 2 containing magnets Rotation mechanism (first magnet rotation mechanism, second magnet rotation mechanism), and a fluorescence imaging processing module, each fluid isolation valve 7 is correspondingly installed with a down lever that controls its switch, and a plunger in each compartment Each 2022 corresponds to the installation of a pressing rod to control its pressing down.
4.当已经包含待测样本的集成化卡盒放置于仪器之后，多个下压杆分别将第一流体隔离阀701、第二流体隔离阀702、第三流体隔离阀703、 第四流体隔离阀704关闭，第一气道接口1071关闭，此时腔室2011便形成了密闭的独立空间。开启外源仪器上位于腔室2011下方的第一磁铁旋转机构，即可实现样本与预埋试剂的充分混合反应。这一步的目的是为了让样本中的待检测核酸释放出来。4. After the integrated cassette containing the sample to be tested is placed in the instrument, a plurality of lower pressure rods separate the first fluid isolation valve 701, the second fluid isolation valve 702, the third fluid isolation valve 703, and the fourth fluid respectively. The valve 704 is closed and the first airway interface 1071 is closed. At this time, the chamber 2011 forms a closed and independent space. Turning on the first magnet rotation mechanism on the external instrument located below the chamber 2011 can achieve a sufficient mixing reaction of the sample and the embedded reagent. The purpose of this step is to release the nucleic acid to be detected in the sample.
5.混合完成后，打开气路接口1071，使得位于第二隔间202a上的下压杆下降，推动柱塞2022压迫隔膜2023膨胀变形，被基片1上的尖锐凸起103刺破，释放出第二隔间202a内的核酸结合液。5. After the mixing is completed, open the gas path interface 1071, so that the lower pressure rod located on the second compartment 202a descends, pushes the plunger 2022 to press the diaphragm 2023 to expand and deform, and is pierced by the sharp protrusion 103 on the substrate 1 and released The nucleic acid binding solution in the second compartment 202a is discharged.
6.保持第一磁铁旋转机构运动，第一磁力转子601将已释放的核酸、磁珠和结合液充分混合，然后打开第一流体隔离阀701，同时气源通过第一气道接口1071对腔室2011内提供正压，推动上述混合液体，按照“腔室2011-第一流体隔离阀701(开启)-核酸固定萃取反应池102-第四流体隔离阀704(关闭)-第五流体隔离阀705(开启)-废液固定腔室203”的顺序进行流动。6. Keep the first magnet rotating mechanism in motion, the first magnetic rotor 601 will fully mix the released nucleic acid, magnetic beads and binding liquid, and then open the first fluid isolation valve 701, and at the same time the gas source passes through the first airway interface 1071 to the cavity Positive pressure is provided in the chamber 2011 to push the above-mentioned mixed liquid, in accordance with the "chamber 2011-first fluid isolation valve 701 (open)-nucleic acid immobilization extraction reaction cell 102-fourth fluid isolation valve 704 (closed)-fifth fluid isolation valve 705 (open)-the waste liquid is flowed in the order of the fixed chamber 203".
7.在此过程中，磁珠颗粒随着液体流动至核酸固定萃取反应池102时，核酸固定萃取反应池102下方的第二磁铁旋转机构保持静止，在磁力的作用下，磁珠颗粒被固定在核酸固定萃取反应池102中，液体则最终进入废液固定腔室203。7. During this process, when the magnetic bead particles flow with the liquid to the nucleic acid fixation extraction reaction cell 102, the second magnet rotating mechanism below the nucleic acid fixation extraction reaction cell 102 remains stationary, and the magnetic bead particles are fixed under the action of magnetic force. In the nucleic acid fixation extraction reaction tank 102, the liquid finally enters the waste liquid fixation chamber 203.
8.然后调整下压杆位置，第一流体隔离阀701关闭，第二流体隔离阀702打开。同时位于第二隔间202b上的下压杆下降，推动柱塞2022压迫隔膜2023膨胀变形，被基片1上的尖锐凸起103刺破，释放出第二隔间202b内的核酸漂洗液。需要特别指出的是，在此过程中，下压杆可以将隔间内的液体一次性压出，也可以通过控制下降高度，定量分批释放液体，进行多次漂洗。8. Then adjust the position of the lower pressing rod, the first fluid isolation valve 701 is closed, and the second fluid isolation valve 702 is opened. At the same time, the lower pressing rod located on the second compartment 202b descends, pushing the plunger 2022 to press the diaphragm 2023 to expand and deform, and is pierced by the sharp protrusion 103 on the substrate 1, releasing the nucleic acid rinsing liquid in the second compartment 202b. It needs to be specially pointed out that in this process, the lower pressing rod can press out the liquid in the compartment at one time, or by controlling the descending height, the liquid can be released quantitatively in batches for multiple rinsing.
9.从第二隔间202b释放的核酸漂洗液通过第二流体隔离阀702进入核酸固定萃取反应池102，核酸固定萃取反应池102为低深宽比的腔室，其投影形状可以是圆形，菱形，橄榄形，葫芦形等，上述各种形状保证液体能将腔室填满。在核酸固定萃取反应池102充满漂洗液后，下方的第二磁铁旋转机构开始运动，带动第二磁力转子602将上一步固定在腔室内的磁珠与漂洗液充分混合。9. The nucleic acid rinsing solution released from the second compartment 202b enters the nucleic acid fixation extraction reaction tank 102 through the second fluid isolation valve 702. The nucleic acid fixation extraction reaction tank 102 is a chamber with a low aspect ratio, and its projection shape can be circular , Diamond-shaped, olive-shaped, gourd-shaped, etc., the above-mentioned various shapes ensure that the liquid can fill the cavity. After the nucleic acid fixation extraction reaction cell 102 is filled with the rinsing liquid, the second magnet rotating mechanism below starts to move, driving the second magnetic rotor 602 to fully mix the magnetic beads fixed in the chamber in the previous step with the rinsing liquid.
10.然后第二磁铁旋转机构停止运动，在磁力的作用下，静置一段时间之后，腔室内悬浮的磁珠颗粒重新被固定在核酸固定萃取反应池102中。10. Then the second magnet rotation mechanism stops moving, and after being allowed to stand for a period of time under the action of magnetic force, the magnetic bead particles suspended in the chamber are fixed in the nucleic acid immobilization extraction reaction cell 102 again.
11.磁珠固定完成后，打开第二气道接口1072和第三流体隔离阀703，在外部气源的作用下，将核酸固定萃取反应池102内的漂洗液推入废液固 定腔室203。11. After the magnetic beads are fixed, open the second airway interface 1072 and the third fluid isolation valve 703, and push the rinsing liquid in the nucleic acid fixation extraction reaction tank 102 into the waste liquid fixing chamber 203 under the action of the external air source .
12.漂洗步骤完成后，保持第三流体隔离阀703打开，位于第三隔间203上的下压杆下降，推动柱塞2032压迫隔膜2033膨胀变形，被基片1上的尖锐凸起103刺破，释放出第三隔间202c内的核酸洗脱液。12. After the rinsing step is completed, keep the third fluid isolation valve 703 open, the lower pressing rod located on the third compartment 203 descends, pushes the plunger 2032 to press the diaphragm 2033 to expand and deform, and is pierced by the sharp protrusion 103 on the substrate 1 The nucleic acid eluate in the third compartment 202c is released.
13.从第三隔间202c释放的核酸洗脱液通过第三流体隔离阀703进入核酸固定萃取反应池102。在核酸固定萃取反应池102充满洗脱液后，下方的第二磁铁旋转机构开始运动，带动第二磁力转子602将上一步固定在腔室内的磁珠与洗脱液充分混合，同时开启位于核酸固定萃取反应池102下方的第一加热模块。“混合+加热”的温度控制在50-80℃，时间控制在180-600s，效果最佳。13. The nucleic acid eluate released from the third compartment 202c enters the nucleic acid fixation extraction reaction cell 102 through the third fluid isolation valve 703. After the nucleic acid fixation and extraction reaction cell 102 is filled with the eluent, the second magnet rotating mechanism below starts to move, driving the second magnetic rotor 602 to fully mix the magnetic beads fixed in the chamber in the previous step with the eluent, and at the same time turn on the nucleic acid The first heating module under the extraction reaction tank 102 is fixed. The temperature of "mixing + heating" is controlled at 50-80℃, and the time is controlled at 180-600s, the effect is the best.
14.完成上述操作后，打开第四流体隔膜阀704，关闭第五流体隔膜阀705，在外部气源的作用下，洗脱液从核酸固定萃取反应池102进入固体试剂存储组件3的存储筒301，并与其中的试剂混合。14. After completing the above operations, open the fourth fluid diaphragm valve 704 and close the fifth fluid diaphragm valve 705. Under the action of an external gas source, the eluent enters the storage cylinder of the solid reagent storage assembly 3 from the nucleic acid immobilization extraction reaction cell 102 301, and mixed with the reagents in it.
15.位于存储筒301上方的下压杆下降，推动混合后的试剂进入扩增反应区4内的反应孔401。15. The lower pressing rod located above the storage cylinder 301 descends, pushing the mixed reagents into the reaction hole 401 in the amplification reaction zone 4.
16.然后通过下压杆下降关闭第六流体隔膜阀706，各个反应孔401与外界隔绝，打开位于扩增反应区4下方的第二加热模块，打开位于扩增反应区4上方的荧光成像处理模块。控制反应温度，各独立反应孔开始进行核酸扩增反应，荧光成像处理模块根据反应孔的光信号，对目标基因进行检测。16. Then the sixth fluid diaphragm valve 706 is closed by the lower pressing rod, each reaction hole 401 is isolated from the outside, the second heating module located below the amplification reaction zone 4 is turned on, and the fluorescence imaging process located above the amplification reaction zone 4 is turned on Module. The reaction temperature is controlled, and each independent reaction hole starts a nucleic acid amplification reaction, and the fluorescence imaging processing module detects the target gene according to the light signal of the reaction hole.
实施例2——集成核酸提取与巢式扩增Example 2-Integrated nucleic acid extraction and nested amplification
1.现将本发明描述的核酸自动化提取并检测的集成化卡盒的另一种实施方式描述如下：1. Now another embodiment of the integrated cassette for automatic nucleic acid extraction and detection described in the present invention is described as follows:
2.该实施例第一阶段操作，与实施例1的步骤1-13基本相同，即将第三隔间202c中的核酸洗脱液注入核酸固定萃取反应池102中，但通过控制所对应柱塞2032的下降距离，使得洗脱液不完全充满核酸固定萃取反应池102；2. The first stage operation of this embodiment is basically the same as steps 1-13 of embodiment 1, that is, the nucleic acid eluate in the third compartment 202c is injected into the nucleic acid fixation extraction reaction cell 102, but the corresponding plunger is controlled The descent distance of 2032 makes the eluate not completely fill the nucleic acid fixation extraction reaction cell 102;
3.核酸洗脱完成后，保持第一流体隔离阀701、第二流体隔离阀702、第三流体隔离阀703、第四流体隔离阀704关闭，第五流体隔离阀705、第七流体隔离阀707打开，下压预扩增试剂隔间202d上方的柱塞2032，释放一定量的预扩增试剂进入核酸固定萃取反应池102，下方的第二磁铁旋转机构运动，带动第二磁力转子602将上一步已经被洗脱下的核酸与预扩增试剂进行充分混合，混合时间5-60s效果最佳。3. After the nucleic acid elution is completed, keep the first fluid isolation valve 701, the second fluid isolation valve 702, the third fluid isolation valve 703, and the fourth fluid isolation valve 704 closed, and the fifth fluid isolation valve 705 and the seventh fluid isolation valve 707 is opened, and the plunger 2032 above the pre-amplification reagent compartment 202d is pressed down to release a certain amount of pre-amplification reagent into the nucleic acid fixed extraction reaction cell 102. The second magnet rotation mechanism below moves, driving the second magnetic rotor 602 to move The nucleic acid that has been eluted in the previous step is thoroughly mixed with the pre-amplification reagent, and the mixing time is 5-60s for the best effect.
4.混合完毕后，关闭流体隔膜阀第一流体隔离阀701、第二流体隔离阀702、第三流体隔离阀703、第四流体隔离阀704、第五流体隔离阀705、第七流体隔离阀707，开启位于核酸固定萃取反应池102下方的第一加热模块，进行核酸的预扩增反应。该扩增反应可以是等温核酸扩增，也可以是变温核酸扩增。4. After mixing, close the first fluid isolation valve 701, the second fluid isolation valve 702, the third fluid isolation valve 703, the fourth fluid isolation valve 704, the fifth fluid isolation valve 705, and the seventh fluid isolation valve. 707. Turn on the first heating module under the nucleic acid fixation extraction reaction cell 102 to perform a nucleic acid pre-amplification reaction. The amplification reaction may be isothermal nucleic acid amplification or variable temperature nucleic acid amplification.
5.预扩增完毕后，打开第五流体隔离阀705、第七流体隔离阀707，通过控制外部气源的流量，使得部分扩增产物通过第四流体隔离阀704、第五流体隔离阀705进入废液固定腔室203。剩余量可以是核酸固定萃取反应池102总体的1/10-1/100，也可以是其他根据实际扩增反应所需要的合适比例。5. After the pre-amplification is completed, open the fifth fluid isolation valve 705 and the seventh fluid isolation valve 707, and by controlling the flow of the external air source, part of the amplified product passes through the fourth fluid isolation valve 704 and the fifth fluid isolation valve 705 Enter the waste liquid fixing chamber 203. The remaining amount can be 1/10-1/100 of the total nucleic acid fixation extraction reaction tank 102, or other suitable ratios required by the actual amplification reaction.
6.打开第三流体隔膜阀703，关闭第四流体隔膜阀704，下压第三隔间202c上方的柱塞2032，释放核酸洗脱液进入并充满核酸固定萃取反应池102，这时核酸洗脱液起到稀释预扩增产物的作用。下方的第二磁铁旋转机构运动，带动第二磁力转子602将上一步的预扩增产物与稀释液进行充分混合，混合时间5-60s效果最佳。6. Open the third fluid diaphragm valve 703, close the fourth fluid diaphragm valve 704, press down the plunger 2032 above the third compartment 202c, and release the nucleic acid eluate into and fill the nucleic acid immobilization extraction reaction cell 102. At this time, the nucleic acid is washed Deliquation plays a role in diluting the pre-amplified product. The movement of the second magnet rotating mechanism below drives the second magnetic rotor 602 to fully mix the pre-amplified product of the previous step with the diluent, and the mixing time is 5-60s for the best effect.
7.完成上述操作后，打开第四流体隔膜阀704，关闭第五流体隔膜阀705，在外部气源的作用下，被稀释的预扩增产物从核酸固定萃取反应池102进入固体试剂存储组件3的存储筒301，并与其中的试剂混合。7. After completing the above operations, open the fourth fluid diaphragm valve 704 and close the fifth fluid diaphragm valve 705. Under the action of an external air source, the diluted pre-amplification product enters the solid reagent storage assembly from the nucleic acid immobilization extraction reaction cell 102 3 of the storage cylinder 301, and mixed with the reagents in it.
8.此后操作与实施例1中15-16步骤基本相同。8. The subsequent operations are basically the same as steps 15-16 in Example 1.
本发明提供的一种核酸检测卡盒，具有以下优点：The nucleic acid detection cartridge provided by the present invention has the following advantages:
1.核酸提取与扩增所需的所有试剂都内置在集成化卡盒(即芯片)上，包括液体试剂、干粉试剂，用户只需要加入样品，操作极其简单，真正适用于基层用户使用。1. All the reagents required for nucleic acid extraction and amplification are built into the integrated cartridge (ie chip), including liquid reagents and dry powder reagents. Users only need to add samples. The operation is extremely simple and it is really suitable for basic users.
2.可以做巢式扩增，大大提高了灵敏度。2. It can do nested amplification, which greatly improves the sensitivity.
3.各组件均采用医疗行业常用材质和工艺，成本降低很多。3. All components are made of materials and processes commonly used in the medical industry, which reduces the cost a lot.
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be considered that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, a number of simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims (10)

  1. 一种核酸检测卡盒，其特征在于：包括基片、液体存储组件、固体试剂存储组件、扩增反应区，所述基片与所述扩增反应区连接，所述液体存储组件、固体试剂存储组件分别设置在所述基片上，所述液体存储组件与所述固体试剂存储组件通过微流道连通，所述固体试剂存储组件与所述扩增反应区通过微流道连通。A nucleic acid detection cartridge, which is characterized in that it comprises a substrate, a liquid storage component, a solid reagent storage component, and an amplification reaction zone, the substrate is connected to the amplification reaction zone, the liquid storage component, and the solid reagent The storage components are respectively arranged on the substrate, the liquid storage component and the solid reagent storage component are communicated with each other through a micro flow channel, and the solid reagent storage component is communicated with the amplification reaction zone through a micro flow channel.
  2. 根据权利要求1所述的核酸检测卡盒，其特征在于：所述液体存储组件包括样本存储组件、提取试剂存储组件、废液固定腔室，所述基片上设有核酸固定萃取反应池，所述样本存储组件、提取试剂存储组件的输出端分别通过微流道与所述核酸固定萃取反应池的输入端连通，所述核酸固定萃取反应池的输出端分别通过微流道与所述固体试剂存储组件、废液固定腔室连通。The nucleic acid detection cartridge according to claim 1, wherein the liquid storage component includes a sample storage component, an extraction reagent storage component, and a waste liquid fixing chamber, and the substrate is provided with a nucleic acid fixing extraction reaction cell, so The output ends of the sample storage component and the extraction reagent storage component are respectively connected to the input end of the nucleic acid immobilization extraction reaction cell through a micro flow channel, and the output ends of the nucleic acid immobilization extraction reaction cell are respectively connected to the solid reagent through the micro flow channel The storage component and the waste liquid fixed chamber are in communication.
  3. 根据权利要求2所述的核酸检测卡盒，其特征在于：所述提取试剂存储组件包括多个独立的隔间，所述隔间包括管壁、柱塞和隔膜，所述柱塞设置在所述管壁的上方，所述隔膜设置在所述管壁的下方，所述管壁、柱塞、隔膜之间围合形成液体试剂存储腔体，所述基片上设有与所述隔间一一对应的尖锐凸起，所述尖锐凸起设置在所述隔膜的下方。The nucleic acid detection cartridge according to claim 2, wherein the extraction reagent storage assembly comprises a plurality of independent compartments, the compartments comprise a tube wall, a plunger and a diaphragm, and the plunger is arranged at the Above the tube wall, the diaphragm is arranged below the tube wall, the tube wall, the plunger, and the diaphragm are enclosed to form a liquid reagent storage cavity, and the substrate is provided with a space that is connected to the compartment. A corresponding sharp protrusion, the sharp protrusion is arranged below the diaphragm.
  4. 根据权利要求3所述的核酸检测卡盒，其特征在于：所述隔间至少包括存储结合液的第一隔间、存储漂洗液的第二隔间和存储洗脱液的第三隔间，所述第一隔间通过微流道与所述样本存储组件连通，所述样本存储组件与所述核酸固定萃取反应池之间的微流道上设有第一流体隔离阀，所述第二隔间与所述核酸固定萃取反应池之间的微流道上设有第二流体隔离阀，所述第三隔间与所述核酸固定萃取反应池之间的微流道上设有第三流体隔离阀，所述核酸固定萃取反应池与所述固体试剂存储组件之间的微流道上设有第四流体隔离阀，所述核酸固定萃取反应池与所述废液固定腔室之间的微流道上设有第五流体隔离阀，所述固体试剂存储组件与所述扩增反应区之间的微流道上设有第六流体隔离阀。The nucleic acid detection cartridge according to claim 3, wherein the compartment includes at least a first compartment for storing the binding solution, a second compartment for storing the rinsing solution, and a third compartment for storing the eluent. The first compartment is in communication with the sample storage component through a micro flow channel, and the micro flow channel between the sample storage component and the nucleic acid immobilization extraction reaction cell is provided with a first fluid isolation valve, and the second compartment A second fluid isolation valve is provided on the micro flow channel between the nucleic acid immobilization and extraction reaction cell, and a third fluid isolation valve is provided on the micro flow channel between the third compartment and the nucleic acid immobilization and extraction reaction cell. A fourth fluid isolation valve is provided on the micro flow channel between the nucleic acid fixation extraction reaction cell and the solid reagent storage assembly, and the micro flow channel between the nucleic acid fixation extraction reaction cell and the waste liquid fixation chamber is provided with a first Five fluid isolation valves, a sixth fluid isolation valve is provided on the micro flow channel between the solid reagent storage component and the amplification reaction zone.
  5. 根据权利要求4所述的核酸检测卡盒，其特征在于：所述隔间还包括存储预扩增试剂的预扩增试剂隔间，所述预扩增试剂隔间通过微流道与所述核酸固定萃取反应池连通，所述预扩增试剂隔间与所述核酸固定萃取反应池之间的微流道上设有第七流体隔离阀。The nucleic acid detection cartridge according to claim 4, wherein the compartment further comprises a pre-amplification reagent compartment for storing pre-amplification reagents, and the pre-amplification reagent compartment is connected to the pre-amplification reagent compartment through the micro channel. The nucleic acid fixation extraction reaction cell is connected, and a seventh fluid isolation valve is provided on the micro flow channel between the pre-amplification reagent compartment and the nucleic acid fixation extraction reaction cell.
  6. 根据权利要求5所述的核酸检测卡盒，其特征在于：所述废液固定腔室 上设有可独立控制开启关闭的第一气道接口和可独立控制开启关闭的第二气道接口，所述样本存储组件与所述第一气道接口连通，所述第三隔间或者预扩增试剂隔间与所述第二气道接口连通。The nucleic acid detection cartridge according to claim 5, wherein the waste liquid fixing chamber is provided with a first airway interface that can be turned on and off independently and a second airway interface that can be turned on and off independently, and The sample storage component is in communication with the first airway interface, and the third compartment or the pre-amplification reagent compartment is in communication with the second airway interface.
  7. 根据权利要求4所述的核酸检测卡盒，其特征在于：所述基片上设有多条相互连通的沟槽，所述基片上贴合有盖片，所述盖片将所述沟槽围合形成微流道，所述基片上设有多个阀座结构，多个所述阀座结构分别安装第一流体隔离阀、第二流体隔离阀、第三流体隔离阀、第四流体隔离阀、第五流体隔离阀、第六流体隔离阀。The nucleic acid detection cartridge according to claim 4, wherein a plurality of interconnected grooves are provided on the substrate, a cover sheet is attached to the substrate, and the cover sheet surrounds the grooves. Combined to form a micro flow channel, the substrate is provided with a plurality of valve seat structures, and the plurality of valve seat structures are respectively installed with a first fluid isolation valve, a second fluid isolation valve, a third fluid isolation valve, and a fourth fluid isolation valve , The fifth fluid isolation valve, the sixth fluid isolation valve.
  8. 根据权利要求2所述的核酸检测卡盒，其特征在于：所述样本存储组件内设有第一磁力转子，所述核酸固定萃取反应池内设有第二磁力转子，所述样本存储组件处安装有驱动所述第一磁力转子转动的第一磁铁旋转机构，所述核酸固定萃取反应池处安装有驱动所述第二磁力转子转动的第二磁铁旋转机构，所述核酸固定萃取反应池处安装有第一加热模块，所述扩增反应区处安装有第二加热模块。The nucleic acid detection cartridge of claim 2, wherein the sample storage assembly is provided with a first magnetic rotor, the nucleic acid immobilization extraction reaction cell is provided with a second magnetic rotor, and the sample storage assembly is installed at There is a first magnet rotating mechanism that drives the first magnetic rotor to rotate, a second magnet rotating mechanism that drives the second magnetic rotor to rotate is installed in the nucleic acid fixed extraction reaction cell, and the nucleic acid fixed extraction reaction cell is installed There is a first heating module, and a second heating module is installed in the amplification reaction zone.
  9. 根据权利要求2所述的核酸检测卡盒，其特征在于：所述样本存储组件主要由腔室和盖子组成，在所述样本存储组件的腔室内部预存有用于核酸提取流程中所需的核酸捕获磁珠，所述废液固定腔室内含多孔吸水材料。The nucleic acid detection cartridge according to claim 2, wherein the sample storage component is mainly composed of a chamber and a lid, and the nucleic acid required in the nucleic acid extraction process is pre-stored in the chamber of the sample storage component. To capture the magnetic beads, the waste liquid fixing chamber contains a porous water-absorbing material.
  10. 根据权利要求1所述的核酸检测卡盒，其特征在于：所述扩增反应区主要由多个反应孔和连接反应孔的管道组成，所述扩增反应区通过连接件与所述基片连接，所述扩增反应区上方安装有荧光成像处理模块。The nucleic acid detection cartridge according to claim 1, wherein the amplification reaction zone is mainly composed of a plurality of reaction holes and a pipe connecting the reaction holes, and the amplification reaction zone is connected to the substrate through a connecting piece. Connected, a fluorescence imaging processing module is installed above the amplification reaction zone.
PCT/CN2020/096723 2020-06-10 2020-06-18 Nucleic acid test kit WO2021248531A1 (en)

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